CN107132217A - Cervical cell warburg effect simple detection reagent and preparation method and application thereof - Google Patents
Cervical cell warburg effect simple detection reagent and preparation method and application thereof Download PDFInfo
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- CN107132217A CN107132217A CN201710329180.1A CN201710329180A CN107132217A CN 107132217 A CN107132217 A CN 107132217A CN 201710329180 A CN201710329180 A CN 201710329180A CN 107132217 A CN107132217 A CN 107132217A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/775—Indicator and selective membrane
Abstract
The invention relates to a cervical cell Wabbler effect simple detection reagent and a preparation method and application thereof. The reagent consists of a reagent A, a reagent B, a reagent C and a reagent D; the reagent A is 4-6% of subtilisin isotonic solution; the reagent B is acetate buffer solution with pH value of 3.5-6.0; the reagent C consists of 0.1-0.25 g/L of 3,3 ', 5, 5' -tetramethylbenzidine solution, 1.0-2.5 mL/L of 6-methoxyquinoline solution and 2-5mL/L of cumyl hydroperoxide solution; the reagent D consists of 0.03-0.07% toluidine blue O solution and 2.5-7.5 mL/L triton X-100 solution. The reagent can be used for qualitatively detecting the content of free ferroprotoporphyrin and active oxygen of cells of cervical exfoliated cells and judging the strength of the warburg effect of the cervical cells.
Description
Technical field
The present invention relates to a kind of simple detection reagent of cervical cell Warburg effect and its preparation method and application, belong to doctor
Learn inspection field.
Background technology
Cervical carcinoma is second " killer " of WomanHealth, and the whole world is often close on 50 Wan Xinfa cases of cervical cancer, seized every year
The life of global 300,000 women.Chinese cervical cancer pathogenesis number newly-increased every year accounts for female reproductive system malignant tumour more than 150,000
The 73-93% of the incidence of disease.The death rate about 50%.Cervical carcinoma has seriously threatened the health and lives of young and middle-aged women in China.Carry
The key of high cancer cure rate is " three is early "（Early discovery, early diagnosis, early treatment）.Evidence suggests if women is in time
Checked, in early detection cervical carcinoma and strangled the probability in the budding stage to increase significantly.As long as cervical carcinoma
" three is early " can be accomplished, its 5 years survival rates nearly reach 100%.In the past few years, the cancer of China rural area two generaI investigation audient only up to 9%, studies carefully it
Reason, a lack of funds, two lack technology.Due to basic public health infrastructure famine, cytology doctor's wretched insufficiency is higher
Expense, it is more objective to find, and interpretation is simple, and economic biomarker becomes a key to manage cervical carcinoma high-risk women
Problem.
Research finds that cervical cell abnormal carbohydrate metabolism occurs in carcinogenesis, and the consumption sugar amount of cancer cell is in normal cervix
Ten times of chrotoplast, but its energy produced is 1/10th of normal cell, and pentose phosphate pathway metabolic patterns
Active, this exception is referred to as Warburg effect.At present, the Warburg effect of cervical cancer cell can be by PET-CT（Positron emission
Computerized tomograph）Detection is obtained.But, PET-CT operation costs are at a relatively high, and once, amount of radiation is equivalent to one for general physical checkup
Individual normal person's radiation of 30 years received in one hour.Therefore, the new reagent tool for being capable of easy accurate detection cervical carcinoma is developed
There is important realistic meaning.
The content of the invention
To solve that PET-CT operation costs are high, there is heavier radiation hazradial bundle to human body and without easy examination detection cervical carcinoma examination
Agent problem, the invention provides a kind of reagent that quick and precisely can be simply detected to cervical cell Warburg effect.
It is a further object of the present invention to provide a kind of preparation method of the simple detection reagent of cervical cell Warburg effect.
It is thin in detection uterine neck that another object of the present invention is to provide a kind of simple detection reagent of cervical cell Warburg effect
The application of born of the same parents' Warburg effect.
To achieve the above object, the present invention is adopted the following technical scheme that.
A kind of simple detection reagent of cervical cell Warburg effect, is made up of reagent A, reagent B, reagent C and reagent D；
Wherein,
Reagent A is 4-6 % subtilopeptidase A isotonic solution；
Reagent B is pH 3.5-6.0 acetate buffer；
Reagent C is by 0.1 g/L-0.25 g/L 3,3 ', 5,5 '-tetramethyl biphenyl amine aqueous solution, 1.0 mL/L-2.5 mL/L 6-
The cumyl hydroperoxide solution composition of methoxy quinoline solution and 2-5mL/L
Reagent D by 0.03-0.07 % blutenes solution, 2.5-7.5 mL/L triton x-100 solution composition.
The reagent A, reagent B, the volume ratio of reagent C and reagent D are 2:1:1:1.
Preferably,
The concentration of the reagent A subtilopeptidase A is 5 %w.t；
The concentration of the reagent B acetate buffers is 2 mol/L, and pH is 5.0；
The TMB concentration of the reagent C is 0.15 g/L, and 6- methoxy quinolines concentration is 1.25
ML/L, cumyl hydroperoxide concentration is 2.5 mL/L；
The blutene concentration of the reagent D is 0.05 %w.t, and triton x-100 concentration is 5 mL/L.
A kind of preparation method of above-mentioned simple detection reagent, comprises the following steps：
（1）The making of reagent A：
Subtilopeptidase A is added into dissolving in isotonic solution and produces reagent A；
（2）Reagent B making：
a）Sodium acetate and glacial acetic acid are dissolved separately in pure water；
b）It is reagent B by two kinds of solution mixings；
（3）The making of reagent C：
a）3,3 ', 5,5 '-tetramethyl benzidine is dissolved in a small amount of absolute ethyl alcohol；
b）Cumyl hydroperoxide and 6- methoxy quinolines are sequentially added into above-mentioned a）Dissolved in solution；
c）Pure water constant volume, produces reagent C；
（4）The making of reagent D：
a）Blutene is dissolved in pure water；
b）Triton x-100 adds above-mentioned a）Dissolved in solution, produce reagent D.
The percent by volume of the absolute ethyl alcohol and reagent C is 30%.
A kind of above-mentioned simple detection reagent comprises the following steps in the application of detection cervical cell Warburg effect：
（1）Epithelium of cervix uteri cast-off cells are placed in reagent A sample liquid is made；
（2）By the centrifugation of above-mentioned sample liquid；
（3）Take step（2）Supernatant, first add reagent B and mix, add reagent C ferrous with colorimetric method for determining cell free
Protoporphyrin content：If not changing color as feminine gender, light blue is weakly positive, and blueness is the positive, and navy blue is strong positive；
（4）Take step（2）Sediment, add reagent D with the active o content of colorimetric method for determining cervical exfoliated cell：If constant
Color（Blueness）For feminine gender, it is the positive that blueness, which is changed into light blue, is become colorless as strong positive.
The operation principle of simple detection reagent of the invention is as follows：
1. the dissolving of cervical cell：Cervical cell is suspended in pretreatment fluid（Reagent A）In turn into cell suspension, cell membrane fractions
Composition is decomposed by subtilopeptidase A, and cell membrane dissociation, the intracellular matter containing cell free ferroprotoporphyrin is dissolved in
In pretreatment fluid, and the organelle for producing reactive oxygen species is suspended in pretreatment fluid, by centrifuge by organelle and other
Cell residue is precipitated.
2. the test philosophy of cell free ferroprotoporphyrin：Cell free ferroprotoporphyrin has similar peroxidase
Effect, possess hydrolysis peroxide（Such as cumyl hydroperoxide）Ability.Cell free ferroprotoporphyrin and peroxidating
Thing reacts, and captures the oxygen atom of peroxide, and combined with oxygen atom；But the ferrous former porphin of cell free combined with oxygen atom
Quinoline is extremely unstable, and immediately taking off oxygen atom passes to hydrogen donor tetramethyl benzidine again, makes the hydrogen supply for being free of color development group originally
Body TMB dehydrogenation, the blue material benzidine blue of generation group containing color development quinonyl.If in sample not
Containing cell free ferroprotoporphyrin, then generate, do not develop the color without benzidine blue therefore；If ferrous former containing cell free in sample
Porphyrin, then have benzidine blue generation, and blueness is gradually presented in reaction solution.The cell free contained in the depth and sample of colour developing is ferrous
Protoporphyrin is directly proportional.In this way, the color of observing response liquid can survey and sample inner cell dissociate ferroprotoporphyrin content.
3. quantitation active oxygen principle：Cell Fenton reactions are that Intramitochondrial hydrogen peroxide reacts with ferrous ion
Produce the reaction of ferric ion, hydroxyl and hydroxy radical.Hydroxy radical is a kind of oxidability very strong free radical, and it holds very much
Oxidizable various organic matters and inorganic matter, oxidation efficiency are high, and reaction rate is fast.It is anti-that with blutene oxidation occurs for hydroxy radical
Should, the color of blutene is changed, so as to predict the content of hydroxy radical indirectly.If containing cell hydroxyl in sample
Free radical, then blutene is oxidized, and the color of reaction solution is thin out, and the cell hydroxyl contained in the depth and sample of color is free
Base is directly proportional.In this way, the color of observing response liquid can survey and sample inner cell hydroxy radical content.
The present invention has advantages below：
The simple detection reagent of the cervical cell Warburg effect of the present invention can accurately, easily and quickly detect that cell free is sub-
The active o content of iron-protoporphyrin content and cell, the Warburg effect of cervical cell glycometabolism is detected with qualitative method.This hair
Bright reagent stability is good, can stablize under air drying environment and preserve more than 2 years.This cervical cell Warburg effect is simple
Detection reagent susceptibility is high, and specificity is good, meets the requirement of screening agent, is suitable for doing the cervical carcinoma screening of big data crowd.
Embodiment
With reference to embodiment, the present invention will be further described, but the present invention is not limited by following embodiments.
It is prepared by the reagent of embodiment 1
（1）The making of reagent A：
5.00 g subtilopeptidase As are added into the % NaCl isotonic solution of 100mL 0.9 dissolving, reagent A is obtained；
（2）Reagent B making：
a）82g sodium acetates are dissolved in 1000mL water, 2 mol/L sodium acetate solutions are obtained；15g glacial acetic acid is dissolved in 200mL water,
Obtain 2 mol/L acetums；
b）Above-mentioned solution is pressed 5:1 ratio is mixed, and produces reagent B.
（3）The making of reagent C：
a）0.15 3,3 ', 5,5 '-tetramethyl benzidines of g are dissolved in 30 mL absolute ethyl alcohols；
b）0.25 mL cumyl hydroperoxides and 0.125 mL 6- methoxy quinolines are sequentially added into above-mentioned a）It is molten in solution
Solution；
c）Pure water is settled to 100mL, produces reagent C；
（4）The making of reagent D：
a）0.05 g blutenes are dissolved in 100 mL pure water；
b）0.5 mL triton x-100s are added into above-mentioned a）Dissolved in solution, produce reagent D.
The application method of the reagent of embodiment 2
（1）With cytobrush gently swipe epithelium of cervix uteri cast-off cells, then cytobrush is placed in 2mL reagent As and is gently agitated for, made
Into sample liquid；
（2）By above-mentioned 3000 revs/min of high speed centrifugation 3min of sample liquid；
（3）Take step（2）Supernatant, first add 1mL reagents B and mix, add 1mL reagent Cs, the observing response at 3 minutes
The color of liquid, thus shows measure cell free ferroprotoporphyrin content：If not changing color as feminine gender, light blue is weakly positive, blue
Color is the positive, and navy blue is strong positive；
（4）1mL reagent Ds are taken to add step（2）Sediment, thus the color of observing response liquid at 3 minutes show that measure is thin
The content of cytoactive oxygen：If nondiscolouring（Blueness）For feminine gender, it is the positive that blueness, which is changed into light blue, is become colorless as strong positive；
Result judgement：Positive positive for the sample cell Warburg effect in above two detection, there is cervical carcinoma in display
Possibility；The one positive is the sample cell Warburg effect probable positive, and display has the risk of cervical carcinoma；Negative is to be somebody's turn to do
Sample cell Warburg effect is negative, and display there is no the risk of cervical carcinoma.
The clinical verification of embodiment 3
1. susceptibility
（1）Specimen origin：100 parts of the Patients with Cervical Cancer cervical exfoliated cell sample that certain gynaecology of medical institutions stores, cervical carcinoma is equal
Made a definite diagnosis through pathological examination.
（2）Operating method：Operated by embodiment 2.
（3）The result：It is shown in Table 1.
The simple detection reagent susceptibility of the cervical cell Warburg effect of table 1
| Detection project | It is positive | It is negative | Probable positive | Susceptibility（%） |
| Cell free ferroprotoporphyrin is detected | 87 | 13 | -- | 87 |
| Cytoactive oxygen is detected | 64 | 36 | -- | 64 |
| Testing result | 64 | 13 | 23 | 87 |
2. specificity
（1）Specimen origin：Non- 100 parts of the Patients with Cervical Cancer cervical exfoliated cell sample that common gynaecology of certain medical institutions stores.It is non-
Cervical carcinoma excludes cervical carcinoma through pathological examination.
（2）Operating method：Operated by embodiment 2.
（3）The result：It is shown in Table 2.
The simple detection reagent specificity of the cervical cell Warburg effect of table 2
| Detection project | It is positive | It is negative | Suspicious feminine gender | Specificity（%） |
| Cell free ferroprotoporphyrin is detected | 17 | 83 | -- | 83 |
| Cytoactive oxygen is detected | 1 | 99 | -- | 99 |
| Testing result | 1 | 83 | 16 | 99 |
3. youden index
Youden index is calculated by following formula：
The youden index of the simple detection reagent of this cervical cell Warburg effect is 0.86.
From above-mentioned data, the cell free ferroprotoporphyrin of the simple detection reagent of this cervical cell Warburg effect is examined
Survey susceptibility high（87%）, cytoactive oxygen detection specificity height（99%）, the two joint-detection can mutually confirm, with good
Diagnostic accordance rate（86%）.The simple detection reagent of this cervical cell Warburg effect meets the requirement of screening agent, is suitable for doing
The cervical carcinoma screening of big data crowd.
Claims (5)
1. a kind of simple detection reagent of cervical cell Warburg effect, it is characterised in that by reagent A, reagent B, reagent C and examination
Agent D is constituted；Wherein,
Reagent A is 4-6 % w.t subtilopeptidase A isotonic solution；
Reagent B is pH 3.6-6.0 acetate buffers；
Reagent C is by 0.1 g/L-0.25 g/L TMB solution, 1.0 mL/L-2.5 mL/L 6-
The cumyl hydroperoxide solution composition of methoxy quinoline solution and 2-5mL/L；
Reagent D by 0.03-0.07 %w.t blutenes solution, 2.5-7.5 mL/L triton x-100 solution composition；
The reagent A, reagent B, the volume ratio of reagent C and reagent D are 2:1:1:1.
2. simple detection reagent according to claim 1, it is characterised in that
The concentration of the reagent A subtilopeptidase A is 5 %w.t
The concentration of the reagent B acetate buffers is 2 mol/L, and pH is 5.0；
The TMB concentration of the reagent C is 0.15 g/L, and 6- methoxy quinolines concentration is 1.25
ML/L, cumyl hydroperoxide concentration is 2.5 mL/L；
The blutene concentration of the reagent D is 0.05 %w.t, and triton x-100 concentration is 5 mL/L.
3. a kind of preparation method of simple detection reagent as claimed in claim 1 or 2, it is characterised in that comprise the following steps：
（1）The making of reagent A：
Subtilopeptidase A is added into dissolving in isotonic solution and produces reagent A；
（2）Reagent B making：
a）Sodium acetate and glacial acetic acid are dissolved separately in pure water；
b）It is reagent B by two kinds of solution mixings；
（3）The making of reagent C：
a）3,3 ', 5,5 '-tetramethyl benzidine is dissolved in a small amount of absolute ethyl alcohol；
b）Cumyl hydroperoxide and 6- methoxy quinolines are sequentially added into above-mentioned a）Dissolved in solution；
c）Pure water constant volume, produces reagent C；
（4）The making of reagent D：
a）Blutene is dissolved in pure water；
b）Triton x-100 adds above-mentioned a）Dissolved in solution, produce reagent D.
4. preparation method according to claim 3, it is characterised in that the percent by volume of absolute ethyl alcohol and reagent B is
30%。
5. a kind of simple detection reagent as claimed in claim 1 or 2 is in the application of detection cervical cell Warburg effect, it is special
Levy and be, comprise the following steps：
（1）Epithelium of cervix uteri cast-off cells are placed in reagent A sample liquid is made；
（2）By the centrifugation of above-mentioned sample liquid；
（3）Take step（2）Supernatant, first add reagent B and mix, add reagent C, it is ferrous with colorimetric method for determining cell free
Protoporphyrin content：If not changing color as feminine gender, light blue is weakly positive, and blueness is the positive, and navy blue is strong positive；
（4）Take step（2）Sediment, add reagent D with the active o content of colorimetric method for determining cervical exfoliated cell：If constant
Color（Blueness）For feminine gender, it is the positive that blueness, which is changed into light blue, is become colorless as strong positive.
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